Atg4b Overexpression Extends Lifespan and Healthspan in Drosophila melanogaster

Autophagy plays important but complex roles in aging, affecting health and longevity. We found that, in the general population, the levels of ATG4B and ATG4D decreased during aging, yet they are upregulated in centenarians, suggesting that overexpression of ATG4 members could be positive for healthspan and lifespan. We therefore analyzed the effect of overexpressing Atg4b (a homolog of human ATG4D) in Drosophila, and found that, indeed, Atg4b overexpression increased resistance to oxidative stress, desiccation stress and fitness as measured by climbing ability. The overexpression induced since mid-life increased lifespan. Transcriptome analysis of Drosophila subjected to desiccation stress revealed that Atg4b overexpression increased stress response pathways. In addition, overexpression of ATG4B delayed cellular senescence, and improved cell proliferation. These results suggest that ATG4B have contributed to a slowdown in cellular senescence, and in Drosophila, Atg4b overexpression may have led to improved healthspan and lifespan by promoting a stronger stress response. Overall, our study suggests that ATG4D and ATG4B have the potential to become targets for health and lifespan interventions.


Introduction
Aging itself is the single most important risk factor for aging-related diseases, ranging from neurodegenerative diseases, cardiovascular diseases, to diabetes and cancer [1]. During the aging process, metabolism, immunity, fitness and many other bodily functions decline [2]. At cellular level, the accumulation of damaged macromolecules and organelles in cells that could not be removed in time due to declining autophagy function accelerate the aging process and increase disease risk [3]. Changes at the molecular level as a result of organismal aging include the upregulation of the mTOR pathway and immune pathway, and downregulation of MAPK, FOXO, lysosomal-autophagic signaling pathway and metabolic pathway [4][5][6]. These alterations in various types of cells in the organism lead to the aging of tissues and, ultimately the organism.

Overexpression of ATG4B Delayed Cellular Senescence
β-galactosidase is a widely used marker of cellular senescence [31] and high expression of P53, P21, P16, IL6, and CXCL10 are molecular markers of cellular senescence [31][32][33][34]. To test whether ATG4 family factors prevent cellular senescence, we overexpressed ATG4B in IMR90 of passage 39 cells, and tested in passage 47 cells (Figure 2A,B), and found that the overexpression reduced β-galactosidase staining in the cells ( Figure 2C). Similarly, reduced transcript levels of cellular senescence-related marker genes P53, P21, P16, IL6, and CXCL10 ( Figure 2D) and reduced P21 protein levels ( Figure 2B) were also observed. This result is consistent with our previous analysis of ATG4D [27], and demonstrated that the overexpression of ATG4B also delay cellular senescence.

Maintaining Early Atg4b Expression Levels during Late Life Extended Drosophila Lifespan
To test whether the overexpression of Atg4b (a homolog of human ATG4D) extends the lifespan in an organism, we analyzed Drosophila Atg4b overexpression in transgenic flies. The F1 generation from da-GS-Gal4 Drosophila crossed with Atg4b transgenic flies were induced by 100 µM RU486 to overexpress Atg4b in whole body, which was confirmed via RT-PCR ( Figure 3A). The F1 generation from Elav-Gal4 Drosophila crossed with Atg4b transgenic flies were induced by 100 µM RU486 to overexpress Atg4b in the nervous system, which was confirmed via RT-PCR ( Figure 3A). Drosophila with systemic overexpression of Atg4b detected elevated levels of Atg8a (a homologue of Human LC3B) protein downstream of Atg4 in the gut ( Figure 3B), indicating that our system was working. Since food intake is a well-known factor affecting lifespan, and as an important control, we measured food intake in these flies, and found that there was no difference between the control and experimental groups ( Figure 3C), therefore, the effects seen in the following experiments was solely due to Atg4b overexpression.
The induction of Atg4b overexpression in whole body at 2 days of age had no effect on lifespan ( Figure 3E). In contrast, the induction of Atg4b overexpression in whole body from mid-life, i.e., at 40, 45, or 50 days of age, prolonged lifespan in Drosophila. All groups of the median lifespan were extended from 57 to 59 days, which is an increase of 3.5% ( Figure 3D). Given the different lifespan outcomes between early induction and mid-life inductions, we examined the expression levels of Atg4b in these groups, and found that the early induction group (induced at day 2, and measured at day 15) has as much as five times of Atg4b than the control group, while the mid-life induction group (induced at day 40, and measured at day 55) has a level similar to younger flies at day 15 ( Figure 3F). This result suggests that excessive amount of Atg4b expression in Drosophila early in life was not beneficial for lifespan extension, while moderate induction since mid-life resulted in lifespan extension.
Since autophagy is also associated with neurodegenerative diseases [35], we tested whether Atg4b overexpression specifically in the Drosophila nervous system also improves lifespan. We performed specific induction of neurological overexpression of Atg4b in Drosophila at 2 days and 30 days of age and found that it prolonged Drosophila lifespan. The median lifespan was extended from 65 to 75 days, an increase of 15.38% (induced at day 2), and thereafter the median lifespan was extended from 65 to 71 days, an increase of 9.23% (induced at day 30) ( Figure 3G). This suggests that CNS is more sensitive to low levels of ATG4 during normal aging and induced Atg4b in the nervous system may enhance autophagy in the brain and thus prolong Drosophila lifespan.

Atg4b Overexpression Improved Drosophila Healthspan
Healthspan measures how long an individual or an organism stays healthy. Factors that increase fitness and stress resistance are important for healthspan and lifespan extension. Important measurement of healthspan in Drosophila is a number of fitness and stress resistance tests include resistance to oxidative stress, desiccation stress and fitness to assess healthspan. Resistance to oxidative stress mainly reflects the DNA repair capacity and ROS metabolism, while the resistance to starvation and desiccation depends on autophagy and regulation of metabolism [36][37][38][39][40][41]. Here, flies with Atg4b overexpression in whole body induced at 2 days old were assayed at 20 days of age, while the flies induced at 40 days of age were assayed at day 55.
Oxidative stress level increases with age and affects the normal function of various tissues, leading to many chronic diseases associated with aging, such as diabetes, cardiovascular and cerebrovascular and skeletal muscle diseases [42,43]. Hydroxyl radicals (OH) are highly reactive towards lipids, proteins and DNA and, at high concentrations, are severely detrimental to cell survival [44]. The resulting OS is an imbalance between the formation of reactive oxygen species (ROS) and antioxidant defense mechanisms. To test whether Atg4b protects flies against oxidative stress, we added 30% hydrogen peroxide (H 2 O 2 ) to the medium, and found that the induction of Atg4b overexpression in Drosophila at both 2 and 40 days of age improved the resistance to oxidative stress. The median survival time increased from 19.42 h to 23.42 h, an increase of 20.6% (induced at day 2, and tested at day 20), and later it increased by 19.16% (induced at day 40, and tested at day 55) ( Figure 4A). RT-PCR detection after oxidative stress treatment was applied for 10 h showed that the expression of anti-oxidative stress genes increased in the Atg4b overexpression group. Specifically, Atg4b enhanced the activation of several genes in the oxygen free radical scavenging pathways, including NOX, GstE1 and gla2, resulting in enhanced antioxidant capacity in Drosophila. ( Figure 4A).
Desiccation stresses the organism with cell dehydration which could damage the phospholipid bilayer of cell membranes [45], concentrate electrolytes and damage the structure of DNA and multiprotein complexes [46,47]. Desiccation survival relays on the proper metabolic and homeostasis regulation. During this test, flies were subjected to desiccation stress at 25 • C by incubating in vials without water and food. Drosophila with induced Atg4b overexpression at 2 days of age had increased desiccation resistance, with the median survival extended from 25.9 h to 32.3 h, an increase of 24.79%, however, unlike the oxidative stress test, Drosophila with Atg4b overexpression at 40 days of age showed no difference in desiccation resistance from the controls ( Figure 4C). Thus, increased expression of Atg4b promoted resistance to desiccation stress during early, but not late lifespan.
From the results of oxidative stress, fitness and desiccation stress experiments, we conclude that overexpressing Atg4b in Drosophila could improve the antioxidant capacity and enhance muscle motility, with the exception that Atg4b overexpression enhances Drosophila desiccation resistance only when induced at an early stage but not later in life.
With the accumulate dysfunctional mitochondria and protein aggregates resulting from aging, muscle function declines, which is manifested in decreased muscle strength and total muscle mass, and this could be measured via fitness tests [12,48]. To test whether Atg4b also improve fitness in flies, we placed the flies in empty culture tubes, tied to a horizontal board together with a ruler, and filmed to record the climbing process to the top of the tube after they were shaken down to the bottom of the tube. Climbing ability was measured by climbing speed. We found that Atg4b overexpression in Drosophila at both early life of 2 days and mid-life from 40 days of age improved the climbing ability of Drosophila. The vertical climbing speed is increased from 1.447 to 1.602 mm/s, an increase of 10.7% (induced at day 2, and tested at day 20), and then it is increased from 0.2837 to 0.3623 mm/s, an increase of about 21.7% (induced at day 40, and tested at day 55) (Figure 4B).
From the results of oxidative stress, fitness and desiccation stress experiments, we conclude that overexpressing Atg4b in Drosophila could improve the antioxidant capacity and enhance muscle motility, with the exception that Atg4b overexpression enhances Drosophila desiccation resistance only when induced at an early stage but not later in life.

Atg4b Regulated Metabolism and Cell Cycle, and Facilitated Switch to Stress Response Pathways
In the above experiments, we observed apparently contrasting results between lifespan and desiccation tests when Atg4b was induced in whole body early in life versus mid-life, i.e., early induction promoted desiccation resistance, yet had no beneficial effects on lifespan, whereas mid-life induction extended lifespan but had no effects on desiccation resistance. In an attempt to explain the apparent contradictory results, we checked the expression of Atg4b at 22 h after desiccation stress was applied, and found that the level of Atg4b in overexpression group (induced at day 2 and tested at day 20) quickly dropped to a lower level than control flies ( Figure 5E), suggesting that the level of ATG4 is subject to rapid down regulation in desiccation stress, and the additional high level of Atg4b, which only exist when induced early, provided this protection from stress. The day 40 induction, in contrast, could not induce as high a level as day 2 induction, and thus did not offer protection in desiccation stress.
Next, we conducted a transcriptome analysis on these flies. We found that in the control group, the 22 h of desiccation treatment increased cell cycle, endosomal transport and protein phosphorylation related genes, while it downregulated organic acid metabolic process, ribosome biogenesis and mitochondrial translation related genes ( Figure 5A). In comparison, in Atg4b overexpression flies, the treatment upregulated cellular response to chemical stimulus, metamorphosis and regulation of intracellular signal transduction, while downregulating mRNA/ncRNA metabolic process, ribosome biogenesis and mitochondrial gene expression ( Figure 5B). These results suggest that Atg4b overexpression increased the activation of stress response pathways, and as a result, may have improved their resistance.
We next compared the overexpression group with controls under these treatment conditions. In 0 h (untreated), overexpressing Atg4b reduced metabolism but increased cell cycle and related transcription pathways ( Figure 5C); when desiccation stress was applied for 22 h, stress response pathways, cell communication pathways increased, while cell cycle and related transcriptional pathways become downregulated ( Figure 5D). As the overexpressed Atg4b level dropped rapidly following the desiccation test, which coincided with decrease in cell cycle related genes, we speculate that the overexpressed Atg4b may have increased cell cycle activity. Consistent with this notion, in IMR90 of passage 47 cells, overexpressing ATG4D indeed enhanced cell activity and accelerated cell proliferation ( Figure 5F). checked the expression of Atg4b at 22 h after desiccation stress was applied, and found that the level of Atg4b in overexpression group (induced at day 2 and tested at day 20) quickly dropped to a lower level than control flies ( Figure 5E), suggesting that the level of ATG4 is subject to rapid down regulation in desiccation stress, and the additional high level of Atg4b, which only exist when induced early, provided this protection from stress. The day 40 induction, in contrast, could not induce as high a level as day 2 induction, and thus did not offer protection in desiccation stress.

Discussion
We studied the ATG4 factors, as ATG4B, and ATG4D were highly expressed in centenarians [27], yet their levels are progressively lower in the general population [28] ( Figure 1A,D). First, we found that overexpression of ATG4D and ATG4B in IMR90 of passage 47 cells can delay cellular senescence (Figure 2, [27]), suggesting that the overexpression of ATG4B andATG4D in centenarians could be a positive factor for longevity, rather than a result of extreme aging.
The p-values in the regression analysis of ATG4B/4D with age were very small despite the low R 2 ( Figure 1A,D), indicating a trend toward significance despite the high variability of the sample data between individuals. In particular, there were very significant differences in the distribution at later stages. Since this dataset was not sampled continuously, we hypothesize that the strong variation in expression at the high end of the horizontal axis (around 80-90 years) may divide the population into two categories: those who continue to live long lives (e.g., centenarians) and those who have reached a late stage of life where high expression of ATG4B and ATG4D may help achieve longevity. In Drosophila, we added 100 µM of RU486 to the control flies and found that it did not affect lifespan (Supplementary Figure S3). The overexpression of Atg4b increased resistance to oxidative stress, desiccation stress and climbing ability (Figure 4), and the overexpression induced since mid-life increased lifespan ( Figure 3D). These suggest that maintaining Atg4b expression levels at mid-late stages of Drosophila life is beneficial for extending lifespan and improving health. It suggests that the reduction in Atg4b due to aging is counterbalanced to achieve a delay in the aging process.
Transcriptome analysis during desiccation stress verified that Atg4b confers resistance to desiccation stress likely by quickly switching from promoting cell cycle to activating stress resistance pathways ( Figure 5). This study suggests that the overexpression of ATG4D in centenarians could be a positive factor for human health and lifespan rather than a consequence of extreme aging.
Activation of the autophagic pathway is beneficial to extend lifespan, but excessive activation may also lead to accelerated aging [17,27], thus, all overexpression autophagy factors are not positive for health and lifespan. Our results show that the timing of overexpression is also essential, as the induction of Atg4b overexpression at 2 days of age has no effect on Drosophila lifespan, but there is a transient phase of substantial increase in mortality throughout mid-life ( Figure 3E). However, the induction of Atg4b overexpression at 40 days of age was able to prolong Drosophila lifespan ( Figure 3D). The higher expression level of Atg4b in the day 2 induction group than in the day 40 group (Figure 3F), suggests that too high a level of Atg4b was not conducive to the extension of lifespan. Additionally, since the expression of Atg4b decreased with age ( Figure 1B), maintaining its level later in life is more important than excessive early expression for lifespan extension.
The lysosomal-autophagic pathway also plays an important role in neurodegenerative diseases [49][50][51][52], as we previously showed that Drosophila dNAGLU (a homolog of human NAGLU, a lysosomal enzyme) reduced Aβ42 deposition in AD Drosophila and human U251-APP cells by enhancing lysosomal function [53]. At the same time, in a recently published paper, it was found that bi-allelic loss-of-function variants in ATG4D is the cause of syndromic neurodevelopmental disorders [54]. Here, we found that specific overexpression of Atg4b in the Drosophila nervous system at both 2 and 30 days of age prolonged lifespan, with the 2-day-old induction group living longer than the 30-day-old induction group (Figure 3G), suggesting that the CNS could be the main tissue where ATG4D exert their functions, and maintaining higher levels of Atg4b expression in the Drosophila nervous system throughout the life cycle is more conducive to lifespan extension than the induced overexpression in the entire body.
Not all members of ATG4 family act the same way during aging. The expression of ATG4B and ATG4D decreased with aging ( Figure 1A,D), while the expression of ATG4A did not significantly correlate with age and ATG4C increased with aging (Supplementary Figure S1A,B). In senescent cells, both ATG4B and ATG4D overexpression delayed cellular senescence (Figure 2) [27], which may be due to the inconsistent splicing activity among various ATG4 proteins [55].
Transcriptome data offered potential mechanism on how Atg4b may promote health and longevity. During desiccation stress, metabolic pathways were attenuated, while stress pathway genes were upregulated in the Atg4b-overexpressing group, suggesting that these flies can better devote resources to combating stress. The autophagy pathway could be activated by the MAPK pathway cascade to regulate cell proliferation, differentiation, and stress adaptation [56]. Damaged stress-activated autophagy pathway can regulate the cell cycle by downregulating P53 [57,58]. It is possible that enhanced cell proliferation by overexpressed ATG4D in senescent cells ( Figure 5F) and the improved desiccation resistance in Drosophila due to overexpressed Atg4b may upregulate cell cycle-related pathways through the activation of autophagy ( Figures 4C and 5), which in turn enhances the ability of cellular health, cell renewal and improves desiccation resistance.
When desiccation was applied, Atg4b-overexpressing Drosophila Atg4b transcript levels dropped to lower levels than the control (Figure 5E), and a dramatic increase in the death of Drosophila (survival percentage) was observed after Atg4b levels dropped, from more than 40% to less than 10% ( Figure 4C). We speculate that Atg4b plays an important role in regulating the on-or off-state transition of life activity processes. Similarly, in oxidative stress test, where the overexpression of ATG4B and ATG4D enhanced cellular resistance to H 2 O 2 in our in vivo experiments (Supplementary Figure S2), the additional Atg4b may also have increased upregulation of stress response pathways, resulting in stronger oxidative stress resistance. This mechanism linking Atg4b and a faster response to stress may be due to an overall improvement in cellular function, as we have shown that ATG4D promotes the cell cycle in senescent cells. However, it could also be due to an immediate, unknown mechanism.
Taken together, our results revealed an important link between ATG4 factors and healthspan and lifespan, and offered ATG4, in particular ATG4D and ATG4B, as potential target for intervention of neurodegenerative diseases. Further study on ATG4 agonist and Atg4b-specific expression in the Drosophila nervous system ( Figure 3G) could allow us to understand how Atg4b agonists could potentially elevate endogenous Atg4b expression to prolong healthspan and lifespan.

Drosophila Stocks and Construction of the Transgenic Flies
For the construction of transgenic flies, we used molecular cloning technology to insert Atg4b into the UAS promoter-driven vector pUAST-attB to generate pUAST-attB-Atg4b plasmid, which was then microinjected into 86F (III) 6110 transgenic strain (gift of Sun Yat-Sen University) with the attP site inserted near 86F. Atg4b transgenic flies were obtained via standard transgenic. The Atg4b transgenic flies were then backcrossed with w 1118 flies for 5 generations to remove the background. The da-GS-Gal4 flies (gift from Sichuan Agricultural University) expressed steroid-activated GAL4 in the whole body. ELAV (gift from Sichuan Agricultural University) flies expressed steroid-activated GAL4 in the nervous system. The w 1118 flies were wild-type flies.

Husbandry and Lifespan Analysis
The larval stage of all the strains of Drosophila were fed standard cornmeal-yeast medium (agar 7. The Atg4b transgenic flies were crossed with driver lines GS-Gal4 and ELAV-Gal4. In the experimental group, the F1 generation flies were fed SY food supplemented with RU486 (w/v, 100 µM; Sigma-Aldrich, St. Louis, MO, USA, CAS 84371-65-3) to induce Atg4b overexpression in the whole body and nervous system. Additionally, in the control group, F1 generation flies were fed with SY food without RU486 but supplemented with the same dose of ethanol.
After hatching, the larvae were mated in standard cornmeal-yeast medium for at least 48 h, and female flies were selected for experimental research. We used Drosophila for lifespan statistics with 10 flies/tube, and each tube contained an appropriate amount of SY food, at least 20 tubes per experimental condition. All stocks were maintained at 25 • C and 60% humidity, 12 h light, and 12 h dark cycle. The flies were transferred to fresh food every 2 days, during which the number of dead and escaped flies was recorded. All lifespan data were analyzed using the log-rank test.

Health Test Assays
A total of 10 flies/tube were used for the test, with at least 20 tubes per experimental condition. For the desiccation test, the flies were placed in an empty tube without medium, and the number of dead flies was counted every 2 h. For the H 2 O 2 oxidative stress test, circular filter paper was placed at the bottom of the culture tube, 50 µL of 6% glucose solution containing 30% H 2 O 2 was added, then the flies were put into the tube, and the number of dead flies was counted every 2 h [59]. All survival data were analyzed using the log-rank test. For the climbing test, 10 flies/tube were used for the test, with at least 10 tubes per experimental condition. We calculated the average speed of flies climbing from the bottom of the tube to the top to evaluate the climbing ability of flies. The climbing data were analyzed using the t-test.

qPCR
RNA was extracted with RNAiso Plus (TaKaRa, Kusatsu, Japan, RNAA00250). Then, RNA was reverse transcribed into cDNA using Prime Script TMRT reagent kit with gDNA Eraser (TaKaRa, Kusatsu, Japan, RR047Q), and qRT-PCR was performed using the Fast Start Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland, 04913850001) and genespecific primers. All the above experimental operations were performed according to the manufacturer's procedures. Gene expression levels were calculated using the comparative C T method. All primers used are listed in (Supplementary Table S1).

ATG4 Expression Analysis
Changes in ATG4 expression with increasing age in the population [28] were simulated using a simple linear regression model. Pearson's correlation test was used to evaluate the association between gene expression and age through the correlation test function in the R platform. The data that support the findings of this study (RNA-seq datasets) are available in the Gene Expression Omnibus (GEO), accession numbers GSE113957 (human fibroblasts), and data on the expression of ATG4B/4D in HFF cells are available in GSE63577.
Cell passage was performed using a Count Star instrument (MS7-H550-Pro, SCILO-GEX) for live cell counting, followed by equal and uniform plate spreading, and all cell experiments were performed after cell passage to the wall.

Cells and Transfection
IMR90 cells was a gift from Xudong Zhao. ATG4B overexpression plasmid was constructed using the pTomo-CMV-loxP-mRFP-loxP-EGFP lenti-viral vector and was then transduced into IMR90 cells. Stably transfected cells were selected via fluorescence screening.

Cell Proliferation Analysis
For CCK-8 assays, after 48 h of treatment, 10 µL CCK-8 solution (Yeasen, Shanghai, China, #40203ES60) was added to each well. The plates were incubated in an incubator for 3 h, and then absorbance at 450 nm was determined.

SA-β-Galactosidase Staining
NC and ATG4B (OE) groups were stained with β-galactosidase after equal amounts of cells were evenly applied to the wall (each group had six replicate experiments). β-Galactosidase staining was performed using a senescence-associated β-galactosidase staining kit (Beyotime, Shanghai, China). Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then incubated overnight at 37 • C in the dark with the working solution containing 0.05 mg/mL X-gal.
We then divided each group of cell plate into four areas, and randomly selected five visual fields for shooting in each area. Next, the proportion of stained cells in the total number of cells were calculated, and then the average value was taken as the final percentage of each experimental stained cell. Approximately 300 cells were photographed per disc, and data from six repeated experiments from two groups were tested using t-tests.

Bioinformatics Analysis
The polyA-enriched RNA-sequencing (RNA-seq) libraries were prepared for sequencing using the Illumina HiSeq 6000 platform. FastQC v0.11.9 (https://github.com/sandrews/FastQC/releases/tag/v0.11.9 accessed on 26 April 2023) was used for quality control of all raw data. The clean data were then aligned to the D. melanogaster genome in UCSC (dm6) using Hisat2 v2.2.1 [60]. The FeatureCounts [61] was used to count reads of genes. DESeq2 [62] for experimental and control groups for differential gene were screened in R. Differentially expressed genes were subjected to Gene Ontology (GO) enrichment analysis using the ClusterProfiler v4.2.1 package [63] and visualized using the "ggplot2" package in R. Pearson's correlation test was used to evaluate the association between gene expression and age samples through the cor.test function in the R platform. Significance was determined a priori at a Benjamini-Hochberg (BH)-corrected p-value of <0.05.

Statistical Analysis
All survival data were analyzed using the log-rank test (Includes: Natural Life Expectancy Statistics, Stress Survival Statistics). The climbing data, QPCR data and β-galactosidase staining data were analyzed using the t-test. DESeq2 for differential gene screening, Significance was determined a priori at a Benjamini-Hochberg (BH)-corrected p-value of <0.05.